RESUMO
Ten-eleven translocation hydroxylases (TET1-3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In neurons, increased 5hmC levels within gene bodies correlate positively with gene expression. The mechanisms controlling TET activity and 5hmC levels are poorly understood. In particular, it is not known how the neuronal TET3 isoform lacking a DNA-binding domain is targeted to the DNA. To identify factors binding to TET3, we screened for proteins that co-precipitate with TET3 from mouse retina and identified the transcriptional repressor REST as a highly enriched TET3-specific interactor. REST was able to enhance TET3 hydroxylase activity after co-expression and overexpression of TET3-activated transcription of REST target genes. Moreover, we found that TET3 also interacts with NSD3 and two other H3K36 methyltransferases and is able to induce H3K36 trimethylation. We propose a mechanism for transcriptional activation in neurons that involves REST-guided targeting of TET3 to the DNA for directed 5hmC generation and NSD3-mediated H3K36 trimethylation.
Assuntos
Citosina/análogos & derivados , Proteínas de Ligação a DNA/genética , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , 5-Metilcitosina/análogos & derivados , Animais , Citosina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Retina/metabolismo , Ativação Transcricional/genéticaRESUMO
Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Pentoxil (Uracila)/análogos & derivados , Proteínas Proto-Oncogênicas/metabolismo , Timina/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Sequência de Bases , Isótopos de Carbono , Montagem e Desmontagem da Cromatina , Cromatografia Líquida , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases , Células-Tronco Embrionárias/citologia , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Oxirredução , Pentoxil (Uracila)/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Espectrometria de Massas por Ionização por Electrospray , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The synthesis of the triphosphates of 5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine and the incorporation of these building blocks into long DNA fragments using the polymerase chain reaction (PCR) are reported. In this way DNA fragments containing multiple hmC, fC, and caC nucleobases are readily accessible.
